Function

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Overview

Binds to the mitochondrial light strand promoter and functions in mitochondrial transcription regulation. Required for accurate and efficient promoter recognition by the mitochondrial RNA polymerase. Promotes transcription initiation from the HSP1 and the light strand promoter by binding immediately upstream of transcriptional start sites. Is able to unwind DNA. Bends the mitochondrial light strand promoter DNA into a U-turn shape via its HMG boxes. Required for maintenance of normal levels of mitochondrial DNA. May play a role in organizing and compacting mitochondrial DNA.

The segregation of mitochondrial DNA (mtDNA) is important for the maintenance and transmission of the genome between generations. Recently, we clarified that human mitochondrial transcription factor A (TFAM) is required for equal distribution and symmetric segregation of mtDNA in cultured cells; however, the molecular mechanism involved is largely unknown. ClpX is an ATPase associated with various cellular activities (AAA+) proteins that localize to the mitochondrial matrix and is suggested to associate with mtDNA. In this study, we found that RNAi-mediated knockdown of ClpX in HeLa cells resulted in enlarged mtDNA nucleoids, which is very similar to that observed in TFAM-knockdown cells in several properties. The expression of TFAM protein was not significantly reduced in ClpX-knockdown cells. However, the enlarged mtDNA nucleoids caused by ClpX-knockdown were suppressed by overexpression of recombinant TFAM and the phenotype was not observed in knockdown with ClpP, a protease subunit of ClpXP. Endogenous ClpX and TFAM exist in close vicinity, and ClpX enhanced DNA-binding activity of TFAM in vitro. These results suggest that human ClpX, a novel mtDNA regulator, maintains mtDNA nucleoid distribution through TFAM function as a chaperone rather than as a protease and its involvement in mtDNA segregation.

Tfam (transcription factor A, mitochondrial), a DNA-binding protein with tandem high-mobility group (HMG)-box domains, has a central role in the expression, maintenance and organization of the mitochondrial genome. It activates transcription from mitochondrial promoters and organizes the mitochondrial genome into nucleoids. Using X-ray crystallography, we show that human Tfam forces promoter DNA to undergo a U-turn, reversing the direction of the DNA helix. Each HMG-box domain wedges into the DNA minor groove to generate two kinks on one face of the DNA. On the opposite face, a positively charged α-helix serves as a platform to facilitate DNA bending. The structural principles underlying DNA bending converge with those of the unrelated HU family proteins, which have analogous architectural roles in organizing bacterial nucleoids. The functional importance of this extreme DNA bending is promoter specific and seems to be related to the orientation of Tfam on the promoters.

Human mitochondrial transcription factor A, TFAM, is essential for mitochondrial DNA packaging and maintenance and also has a crucial role in transcription. Crystallographic analysis of TFAM in complex with an oligonucleotide containing the mitochondrial light strand promoter (LSP) revealed two high-mobility group (HMG) protein domains that, through different DNA recognition properties, intercalate residues at two inverted DNA motifs. This induced an overall DNA bend of ~180°, stabilized by the interdomain linker. This U-turn allows the TFAM C-terminal tail, which recruits the transcription machinery, to approach the initiation site, despite contacting a distant DNA sequence. We also ascertained that structured protein regions contacting DNA in the crystal were highly flexible in solution in the absence of DNA. Our data suggest that TFAM bends LSP to create an optimal DNA arrangement for transcriptional initiation while facilitating DNA compaction elsewhere in the genome.

Human mitochondrial transcription is driven by a single subunit RNA polymerase and a set of basal transcription factors. The development of a recombinant in vitro transcription system has allowed for a detailed molecular characterization of the individual components and their contribution to transcription initiation. We found that TFAM and TFB2M act synergistically and increase transcription efficiency 100-200-fold as compared with RNA polymerase alone. Both the light-strand promoter (LSP) and the HSP1 promoters displayed maximal levels of in vitro transcription when TFAM was present in an amount equimolar to the DNA template. Importantly, we did not detect any significant transcription activity in the presence of the TFB2M paralog, TFB1M, or when templates containing the putative HSP2 promoter were used. These data confirm previous observations that TFB1M does not function as a bona fide transcription factor and raise questions as to whether HSP2 serves as a functional promoter in vivo. In addition, we did not detect transcription stimulation by the ribosomal protein MRPL12. Thus, only two essential initiation factors, TFAM and TFB2M, and two promoters, LSP and HSP1, are required to drive transcription of the mitochondrial genome.

The mitochondrial transcription factor A (mtTFA) is central to assembly and initiation of the mitochondrial transcription complex. Human mtTFA (h-mtTFA) is a dual high mobility group box (HMGB) protein that binds site-specifically to the mitochondrial genome and demarcates the promoters for recruitment of h-mtTFB1, h-mtTFB2 and the mitochondrial RNA polymerase. The stoichiometry of h-mtTFA was found to be a monomer in the absence of DNA, whereas it formed a dimer in the complex with the light strand promoter (LSP) DNA. Each of the HMG boxes and the C-terminal tail were evaluated for their ability to bind to the LSP DNA. Removal of the C-terminal tail only slightly decreased nonsequence specific DNA binding, and box A, but not box B, was capable of binding to the LSP DNA. The X-ray crystal structure of h-mtTFA box B, at 1.35 A resolution, revealed the features of a noncanonical HMG box. Interactions of box B with other regions of h-mtTFA were observed. Together, these results provide an explanation for the unusual DNA-binding properties of box B and suggest possible roles for this domain in transcription complex assembly.

Mitochondrial transcription factor 1 (mtTF1) is the only accessory protein known to be required for accurate and efficient promoter recognition by mammalian mitochondrial RNA polymerase. It activates transcription by binding immediately upstream of transcriptional start sites and shows an inherent flexibility in primary DNA sequence requirement. By application of a purification strategy designed for human and mouse mtTF1, a protein resembling mtTF1 was recently isolated from yeast mitochondria; its size (19 kDa), DNA-binding properties, and amino acid composition suggest identity to HM, a previously described abundant protein of yeast mitochondria. Both human and yeast proteins show a general ability to wrap or condense and unwind DNA in vitro and bend DNA at specific sequences. Recent determinations of the amino acid sequences of the human and yeast proteins reveal that both contain domains homologous to the nuclear high mobility group (HMG) proteins which have been implicated in diverse functions such as chromatin compaction and transcription stimulation. The ability to unwind and bend DNA may be fundamental to the documented roles of the mammalian protein in mitochondrial DNA transcription and replication priming and suggests a similar function for the yeast protein in yeast mitochondria.

Mitochondrial DNA (mtDNA) occurs in cells in nucleoids containing several copies of the genome. Previous studies have identified proteins associated with these large DNA structures when they are biochemically purified by sedimentation and immunoaffinity chromatography. In this study, formaldehyde cross-linking was performed to determine which nucleoid proteins are in close contact with the mtDNA. A set of core nucleoid proteins is found in both native and cross-linked nucleoids, including 13 proteins with known roles in mtDNA transactions. Several other metabolic proteins and chaperones identified in native nucleoids, including ATAD3, were not observed to cross-link to mtDNA. Additional immunofluorescence and protease susceptibility studies showed that an N-terminal domain of ATAD3 previously proposed to bind to the mtDNA D-loop is directed away from the mitochondrial matrix, so it is unlikely to interact with mtDNA in vivo. These results are discussed in relation to a model for a layered structure of mtDNA nucleoids in which replication and transcription occur in the central core, whereas translation and complex assembly may occur in the peripheral region.

The activity of binding selectively and non-covalently to and distorting the original structure of DNA, typically a straight helix, into a bend, or increasing the bend if the original structure was intrinsically bent due to its sequence.

Tfam (transcription factor A, mitochondrial), a DNA-binding protein with tandem high-mobility group (HMG)-box domains, has a central role in the expression, maintenance and organization of the mitochondrial genome. It activates transcription from mitochondrial promoters and organizes the mitochondrial genome into nucleoids. Using X-ray crystallography, we show that human Tfam forces promoter DNA to undergo a U-turn, reversing the direction of the DNA helix. Each HMG-box domain wedges into the DNA minor groove to generate two kinks on one face of the DNA. On the opposite face, a positively charged α-helix serves as a platform to facilitate DNA bending. The structural principles underlying DNA bending converge with those of the unrelated HU family proteins, which have analogous architectural roles in organizing bacterial nucleoids. The functional importance of this extreme DNA bending is promoter specific and seems to be related to the orientation of Tfam on the promoters.

The mitochondrial transcription factor A (mtTFA) is central to assembly and initiation of the mitochondrial transcription complex. Human mtTFA (h-mtTFA) is a dual high mobility group box (HMGB) protein that binds site-specifically to the mitochondrial genome and demarcates the promoters for recruitment of h-mtTFB1, h-mtTFB2 and the mitochondrial RNA polymerase. The stoichiometry of h-mtTFA was found to be a monomer in the absence of DNA, whereas it formed a dimer in the complex with the light strand promoter (LSP) DNA. Each of the HMG boxes and the C-terminal tail were evaluated for their ability to bind to the LSP DNA. Removal of the C-terminal tail only slightly decreased nonsequence specific DNA binding, and box A, but not box B, was capable of binding to the LSP DNA. The X-ray crystal structure of h-mtTFA box B, at 1.35 A resolution, revealed the features of a noncanonical HMG box. Interactions of box B with other regions of h-mtTFA were observed. Together, these results provide an explanation for the unusual DNA-binding properties of box B and suggest possible roles for this domain in transcription complex assembly.

RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and comprehensive approach, termed "interactome capture," to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.

Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation, and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. To our knowledge, nearly one-third were not previously annotated as RNA binding, and about 15% were not predictable by computational methods to interact with RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of mRNA binders with diverse molecular functions participating in combinatorial posttranscriptional gene-expression networks.

Just as reference genome sequences revolutionized human genetics, reference maps of interactome networks will be critical to fully understand genotype-phenotype relationships. Here, we describe a systematic map of ?14,000 high-quality human binary protein-protein interactions. At equal quality, this map is ?30% larger than what is available from small-scale studies published in the literature in the last few decades. While currently available information is highly biased and only covers a relatively small portion of the proteome, our systematic map appears strikingly more homogeneous, revealing a "broader" human interactome network than currently appreciated. The map also uncovers significant interconnectivity between known and candidate cancer gene products, providing unbiased evidence for an expanded functional cancer landscape, while demonstrating how high-quality interactome models will help "connect the dots" of the genomic revolution.

A significant advancement in understanding mitochondrial gene expression is the recent identification of two new human mitochondrial transcription factors, h-mtTFB1 and h-mtTFB2. Both proteins stimulate transcription in collaboration with the high-mobility group box transcription factor, h-mtTFA, and are homologous to rRNA methyltransferases. In fact, the dual-function nature of h-mtTFB1 was recently demonstrated by its ability to methylate a conserved rRNA substrate. Here, we demonstrate that h-mtTFB1 binds h-mtTFA both in HeLa cell mitochondrial extracts and in direct-binding assays via an interaction that requires the C-terminal tail of h-mtTFA, a region necessary for transcriptional activation. In addition, point mutations in conserved methyltransferase motifs of h-mtTFB1 revealed that it stimulates transcription in vitro independently of S-adenosylmethionine binding and rRNA methyltransferase activity. Furthermore, one mutation (G65A) eliminated the ability of h-mtTFB1 to bind DNA yet did not affect transcriptional activation. These results, coupled with the observation that h-mtTFB1 and human mitochondrial RNA (h-mtRNA) polymerase can also be coimmunoprecipitated, lead us to propose a model in which h-mtTFA demarcates mitochondrial promoter locations and where h-mtTFB proteins bridge an interaction between the C-terminal tail of h-mtTFA and mtRNA polymerase to facilitate specific initiation of transcription. Altogether, these data provide important new insight into the mechanism of transcription initiation in human mitochondria and indicate that the dual functions of h-mtTFB1 can be separated.

Tfam (transcription factor A, mitochondrial), a DNA-binding protein with tandem high-mobility group (HMG)-box domains, has a central role in the expression, maintenance and organization of the mitochondrial genome. It activates transcription from mitochondrial promoters and organizes the mitochondrial genome into nucleoids. Using X-ray crystallography, we show that human Tfam forces promoter DNA to undergo a U-turn, reversing the direction of the DNA helix. Each HMG-box domain wedges into the DNA minor groove to generate two kinks on one face of the DNA. On the opposite face, a positively charged α-helix serves as a platform to facilitate DNA bending. The structural principles underlying DNA bending converge with those of the unrelated HU family proteins, which have analogous architectural roles in organizing bacterial nucleoids. The functional importance of this extreme DNA bending is promoter specific and seems to be related to the orientation of Tfam on the promoters.

Interacting selectively and non-covalently with a sequence of DNA that is in cis with and relatively close to a core promoter for RNA polymerase II (RNAP II) in order to activate or increase the frequency, rate or extent of transcription from the RNAP II promoter.

Interacting selectively and non-covalently with a specific DNA sequence in order to modulate transcription. The transcription factor may or may not also interact selectively with a protein or macromolecular complex.

Tfam (transcription factor A, mitochondrial), a DNA-binding protein with tandem high-mobility group (HMG)-box domains, has a central role in the expression, maintenance and organization of the mitochondrial genome. It activates transcription from mitochondrial promoters and organizes the mitochondrial genome into nucleoids. Using X-ray crystallography, we show that human Tfam forces promoter DNA to undergo a U-turn, reversing the direction of the DNA helix. Each HMG-box domain wedges into the DNA minor groove to generate two kinks on one face of the DNA. On the opposite face, a positively charged α-helix serves as a platform to facilitate DNA bending. The structural principles underlying DNA bending converge with those of the unrelated HU family proteins, which have analogous architectural roles in organizing bacterial nucleoids. The functional importance of this extreme DNA bending is promoter specific and seems to be related to the orientation of Tfam on the promoters.

Dynamic structural changes to eukaryotic chromatin occurring throughout the cell division cycle. These changes range from the local changes necessary for transcriptional regulation to global changes necessary for chromosome segregation.

Mitochondrial transcriptional factor 1 (mtTF1) is required for both transcription and replication of mammalian mitochondrial DNA (mtDNA) and it has two consensus sequences of HMG (high mobility group) boxes. In studies on the regulation of gene expression of mtTF1, we examined the steady state level of the mRNA in cultured HeLa cells. We found that in addition to the major mRNA, 30% of the mRNA of mtTF1 in the cells was a smaller isoform with a 96 base deletion. This smaller mRNA was also found in most human tissues. The region of the deletion corresponds to the second HMG-box, which may interact directly with DNA. We examined the structure of the genomic gene encoding the human mtTF1 to determine the mechanism of the deletion. We found that the gene is composed of 7 exons spanning over 10 kilobase-pairs and that its 5th exon is identical to the 96 bases skipped in the shorter mRNA. Therefore, the shorter mtTF1 is concluded to be generated by alternative splicing.

Human mitochondrial transcription is driven by a single subunit RNA polymerase and a set of basal transcription factors. The development of a recombinant in vitro transcription system has allowed for a detailed molecular characterization of the individual components and their contribution to transcription initiation. We found that TFAM and TFB2M act synergistically and increase transcription efficiency 100-200-fold as compared with RNA polymerase alone. Both the light-strand promoter (LSP) and the HSP1 promoters displayed maximal levels of in vitro transcription when TFAM was present in an amount equimolar to the DNA template. Importantly, we did not detect any significant transcription activity in the presence of the TFB2M paralog, TFB1M, or when templates containing the putative HSP2 promoter were used. These data confirm previous observations that TFB1M does not function as a bona fide transcription factor and raise questions as to whether HSP2 serves as a functional promoter in vivo. In addition, we did not detect transcription stimulation by the ribosomal protein MRPL12. Thus, only two essential initiation factors, TFAM and TFB2M, and two promoters, LSP and HSP1, are required to drive transcription of the mitochondrial genome.

Human mitochondrial transcription is driven by a single subunit RNA polymerase and a set of basal transcription factors. The development of a recombinant in vitro transcription system has allowed for a detailed molecular characterization of the individual components and their contribution to transcription initiation. We found that TFAM and TFB2M act synergistically and increase transcription efficiency 100-200-fold as compared with RNA polymerase alone. Both the light-strand promoter (LSP) and the HSP1 promoters displayed maximal levels of in vitro transcription when TFAM was present in an amount equimolar to the DNA template. Importantly, we did not detect any significant transcription activity in the presence of the TFB2M paralog, TFB1M, or when templates containing the putative HSP2 promoter were used. These data confirm previous observations that TFB1M does not function as a bona fide transcription factor and raise questions as to whether HSP2 serves as a functional promoter in vivo. In addition, we did not detect transcription stimulation by the ribosomal protein MRPL12. Thus, only two essential initiation factors, TFAM and TFB2M, and two promoters, LSP and HSP1, are required to drive transcription of the mitochondrial genome.

Mitochondrial transcriptional factor 1 (mtTF1) is required for both transcription and replication of mammalian mitochondrial DNA (mtDNA) and it has two consensus sequences of HMG (high mobility group) boxes. In studies on the regulation of gene expression of mtTF1, we examined the steady state level of the mRNA in cultured HeLa cells. We found that in addition to the major mRNA, 30% of the mRNA of mtTF1 in the cells was a smaller isoform with a 96 base deletion. This smaller mRNA was also found in most human tissues. The region of the deletion corresponds to the second HMG-box, which may interact directly with DNA. We examined the structure of the genomic gene encoding the human mtTF1 to determine the mechanism of the deletion. We found that the gene is composed of 7 exons spanning over 10 kilobase-pairs and that its 5th exon is identical to the 96 bases skipped in the shorter mRNA. Therefore, the shorter mtTF1 is concluded to be generated by alternative splicing.

Tfam (transcription factor A, mitochondrial), a DNA-binding protein with tandem high-mobility group (HMG)-box domains, has a central role in the expression, maintenance and organization of the mitochondrial genome. It activates transcription from mitochondrial promoters and organizes the mitochondrial genome into nucleoids. Using X-ray crystallography, we show that human Tfam forces promoter DNA to undergo a U-turn, reversing the direction of the DNA helix. Each HMG-box domain wedges into the DNA minor groove to generate two kinks on one face of the DNA. On the opposite face, a positively charged α-helix serves as a platform to facilitate DNA bending. The structural principles underlying DNA bending converge with those of the unrelated HU family proteins, which have analogous architectural roles in organizing bacterial nucleoids. The functional importance of this extreme DNA bending is promoter specific and seems to be related to the orientation of Tfam on the promoters.

Human mitochondrial transcription is driven by a single subunit RNA polymerase and a set of basal transcription factors. The development of a recombinant in vitro transcription system has allowed for a detailed molecular characterization of the individual components and their contribution to transcription initiation. We found that TFAM and TFB2M act synergistically and increase transcription efficiency 100-200-fold as compared with RNA polymerase alone. Both the light-strand promoter (LSP) and the HSP1 promoters displayed maximal levels of in vitro transcription when TFAM was present in an amount equimolar to the DNA template. Importantly, we did not detect any significant transcription activity in the presence of the TFB2M paralog, TFB1M, or when templates containing the putative HSP2 promoter were used. These data confirm previous observations that TFB1M does not function as a bona fide transcription factor and raise questions as to whether HSP2 serves as a functional promoter in vivo. In addition, we did not detect transcription stimulation by the ribosomal protein MRPL12. Thus, only two essential initiation factors, TFAM and TFB2M, and two promoters, LSP and HSP1, are required to drive transcription of the mitochondrial genome.

Keywords

Protein involved in the transfer of genetic information from DNA to messenger RNA (mRNA) by DNA-directed RNA polymerase. In the case of some RNA viruses, protein involved in the transfer of genetic information from RNA to messenger RNA (mRNA) by RNA-directed RNA polymerase.

Protein which is part of a reference proteome. Reference proteomes are a subset of proteomes that have been selected either manually or algorithmically according to a number of criteria to provide a broad coverage of the tree of life and a representative cross-section of the taxonomic diversity found within UniProtKB, as well as the proteomes of well-studied model organisms and other species of interest for biomedical research.